The Y-located locus, testis determining factor (TDF) in humans and testis determining Y (Tdy) in other mammals, is responsible for switching on the testis differentiation. TDF/Tdy requires the interactions of other co- factors to complete this differentiation process. The isolation of the candidate gene, sex determining region Y (SRY/Sry) for TDF/Tdy has provided an excellent opportunity to further define the mechanism by which the SRY/Sry and other testis determining genes mediate sex determination. Recently, we have identified a polymorphism within the glutamine-rich domain of the Sry alleles of several domesticus mouse strains that correlates with the degrees of sex reversal in XY animals when their Y chromosome is introduced into an inbred strain, C57BL/6J (B6), genomic background. We hypothesize that this glutamine-rich domain is involved in a protein-protein interaction between Sry and another co- factor, Sry interactive protein (Sip), to form a transcriptional complex essential for the biological function of Sry. The polymorphism affects such an interaction between the domesticus Sry and the B6 Sip, thereby causing B6.Y/Dom sex reversal. Using a labeled fusion protein containing the Sry-glutamine-rich (Sry-glu) domain as a probe in farwestern blotting we have indeed detected a specific binding between the Sry-glu domain and a protein of about 110 kD in testis extracts. Three achievable specific aims are proposed in the present application to test this hypothesis of sex determination. First, we will correlate the genotypes and phenotypes in B6.Y/Dom animals using gene targeting in embryonic stem (ES) cells and chimeric mouse construction strategies. In these studies, the Sry coding sequence of B6 or 129sv (of the same Sry allele) ES cells will be replaced with those from several domesticus strains that manifest various degrees of B6.Y/dom sex reversal. Their effects in the sex determination of the resulting chimeric mice will be evaluated and correlated to their respective genotypes. Second, we will use several in vitro and in vivo protein-protein interaction screening approaches to isolate the Sip cDNAs from expression cDNA libraries in prokaryotic or yeast cloning systems. The isolated Sip cDNAs will be characterized in terms of their DNA and protein sequences, chromosomal locations, phylogenetic conservation and interaction with Sry. Third, the function of Sip is further evaluated by expression studies in fetal and adult mice and reporter gene assays on its interaction with Sry in transcription regulation. Gene knockout experiments will be performed to define its exact relationship to Sry and sex determination. The proposed studies will provide significant insights on the molecular mechanisms of mammalian sex determination and differentiation and invaluable information on the etiology and clinical management of gonadal dysgenesis and infertility in man.